Malaria proteins localized on the surface of the merozoite has been a major target for developing a much needed multi-subunit vaccine for Plasmodium falciparum malaria, which claims over one million lives each year. The immune response to these merozoite proteins is expected to block parasite invasion into host red blood cells (RBCs). A sialic acid-dependent invasion pathway largely influenced by the interaction of parasite EBA-175 (175 kDa erythrocyte-binding antigen) with sialic acid residues of RBC glycophorin A has been well characterized. However, lines of evidence indicate that this invasion pathway is non-essential for parasite survival. Recently, we identified a sialic acid-independent invasion pathway that relies on the binding of P. falciparum merozoite surface protein-1 (MSP1) to RBC band 3. Here, we propose to extend our findings under the following specific aims. (1) Specificity of the MSPl-band 3 interaction. We propose to map the band 3-binding sites within MSP1, determine binding affinity, and examine the effect of MSP1 domains containing the band 3-binding site on P. falciparum invasion into RBCs. (2) Participation of EBA-175 in the sialic acid-independent invasion pathway. Our preliminary evidence indicates that EBA-175 interacts directly with the band 3 receptor and MSP1, presumably by forming a ternary complex, band 3/MSP1/EBA-175. We propose to investigate a potential role of EBA-175 in the band 3-dependent invasion pathway by characterizing the binding interface of the ternary protein complex. Major methods used will include yeast two-hybrid assay, solution-binding assay using BIACORE biosensor, and site-directed mutagenesis. (3) Role of the band 3 receptor in sialic acid-dependent pathway. We propose to examine the invasion inhibition potential of band 3 in the sialic acid-dependent P. falciparum line maintained in vitro. This study will provide the first evidence on whether the band 3 provides a pathway alternative to the sialic acid-dependent pathway or functions as a primary invasion receptor across P. falciparum lines. Together, a clear understanding of the sialic acid-independent invasion mechanism will provide valuable insights into the development of an effective subunit malaria vaccine.